Download Laurence Lafanechère

Laurence Lafanechère Laurence.lafanechere@ujf-­‐grenoble.fr Microtubules and actin microfilaments are involved in key cell functions!
Mitosis Mo8lity Tubulin Ac8n Polarity & Plas8city Maintenance of shape Perturbation of the regulation of the cytoskeleton can lead to diseases!
Mitosis Mo8lity Cancer Metastasis Polarity & Plas8city Maintenance of shape Neurologic diseases (Schizophrenia, Williams-­‐Beuren syndrome…) Thrombosis Our aims!
ü  To iden8fy crucial cytoskeleton actors deregulated in these diseases ü  To find pharmacological agents that target these proteins ü  To test their therapeu8c efficacy, on in vitro cell models, on animal models and on ex vivo human samples. Post-translational modifications : a mechanism of regulation of the
cytoskeleton functions!
PTMs of proteins that cons8tute the cytoskeleton, for instance tubulin PTMs of associated proteins, for instance cofilin phosphoryla8on SSH LIMK Post-translational modifications : a mechanism of regulation of the
cytoskeleton functions!
PTMs of proteins that cons8tute the cytoskeleton, for instance tubulin PTMs of associated proteins, for instance cofilin phosphoryla8on α-tubulin: ---Gly-Glu-Glu- Tyr
SSH TTL
TCP
α-tubulin:---Gly-Glu-Glu LIMK Post-translational modifications : a mechanism of regulation of the
cytoskeleton functions!
PTMs of proteins that cons8tute the cytoskeleton, for instance tubulin PTMs of associated proteins, for instance cofilin phosphoryla8on α-tubulin : ---Lys40--SSH HDAC6
αTAT1
α-tubulin : ---Lys
--| 40
C=O | CH3 LIMK Methodology and specific tools:!
Different systems to study different functions!
Adhesion, shape change, spreading Migra8on, prolifera8on Cell lines 3D Blood platelets J.
E.
Italiano
et
al.,
1999,
JCB,
147
megacaryocyte platelets 2D nucleus XenograUs on mice Access to specific deficient mice Human ex-­‐vivo samples of cancer cells (blasts, pleural effusions) Methodology and specific tools:!
Specific small molecules discovered by phenotypic screening!
Small molecules that diffuse rapidly into the cytoplasm are valuable probes to study dynamic biological processes Chemical library Robo;c screening on cells Target iden;fica;on Read out : phenotypic change Selec;on of ac;ve compounds Swinney & Anthony, How were new medicines discovered? Nature Review Drug Discovery, 2011
Search for new microtubule regulators, using a phenotypic screen!
Stable
Dynamic
α-tubulin: ---Gly-Glu-Glu- Tyr
1) A cell based assay to select:
- microtubule stabilizing agents
- microtubule
agents
TTL depolymerizing
TCP
Vassal et al., J. Biomol. Screen., 2006
α-tubulin:---Gly-Glu-Glu Tyr-Microtubules
Glu-Microtubules
2) Automated screening of 30,880
chemical compounds, using Taxol®
and colchicine as controls
3) Selection of a stabilizing compound
with novel properties
Pyridocarbazole, Pyr1
85 confirmed
“depolymerizing”
compounds
20 confirmed
“stabilizing”
compounds
-­‐ Stabilizes microtubules and affects ac;n microfilaments -­‐ Without ac;ng directly on tubulin or ac8n -­‐ Impedes cell mo8lity -­‐ Poten8al an;cancer and an;metasta;c agent Patent WO201095042A2, na6onal phases Pyr1 does not act directly on tubulin or actin :
what is its target?!
Chemical library Robo;c screening on cells Target iden;fica;on Read out : phenotypic change Selec;on of ac;ve compounds We were like Prince Charming, in Cinderella, …
… with only a tiny glass slipper,
Pyridocarbazole, to find our beautiful
phenotype.
Hoping that this compound is not like these ugly French
slippers, which fit all feet, including the ugly step sisters’ feet!
Identification of the target(s)
Ø  Using ligand based methods •  Structure/ac8vity rela8onships, to know where it is possible to modify the molecule •  Affinity columns with the compounds / isola8on from cell extracts /
iden8fica8on using proteomics Coll. C-­‐H Nguyen (Ins6tut Curie), plateforme de protéomique (CEA) Subs8tu8on tolerated Crucial for ac8vity To confirme (few analogues) 12 analogues assayed Guess : the target is a kinase…!
Effect of Pyr1 on: •  67 cytoskeleton related kinases •  45 addi8onal diverse kinases (collabora6on with S. Knapp, Oxford University) Pyr1 is a selective LIMK Inhibitor!
30,080 molecules Cell-­‐based assay for microtubule stability increase of Detyr-­‐tubulin Selec;on of pyridocarbazolone LIMK iden;fica;on LIMK
LIM-­‐Pyr1 Inhibition of only one
kinase out of 110 kinases
tested!
Prudent et al., Cancer Res., 2012
LIMK : a signaling node that controls both actin and microtubules dynamics!
TGFβ
VEGF EGF Receptor Cdc42 Rac Rho Cdc42 Pak 1, 2 & 4 ROCK I & II MRCKα
? LIMK Cofilin Cofilin Microtubules dynamics Ac8n dynamics Stabilized microtubules Severed microfilaments P
LIMK: an Emerging Target for Cancer
Chemotherapy
An increasing number of publications confirm that LIM Kinase is a target for cancer therapy
Oncotarget, 2012 LIM-Pyr1 : An “hybrid” mode of action!
Microtubules
Targeting
Agents
Erlotinib
Vinca alkaloids
Taxanes
Epothilones
LIM-Pyr1
Nilotinib
Imatinib
Gefitinib
Sorafenib
Crizotinib
Kinase
Inhibitors
Ongoing studies:!
Fundamental questions!
Ø  Microtubule mechanism of ac8on of LIMK inhibi8on LIMK ? Cofilin Cofilin Microtubules dynamics Ac8n dynamics Ø  Role of LIMK in cell adhesion, spreading, migra8on, invasion T=12h T=0 DMSO LIM-­‐Pyr1 25 μM P
How to bridge the gap between academic research and
clinical application ?!
How to bridge the gap between academic research and
clinical application ?!
ICBS2014 San Francisco How to bridge the gap between academic research and
clinical application ?!
Cell to drug discovery
hWp://cellipse.com/ •  Chemical optimization of LIMK inhibitors
•  Pursuing GLP compliant preclinical development
ICBS2014 San Francisco Acknowledgments!
Biology
Renaud Prudent (IAB, Grenoble)
Anne Martinez (IAB, Grenoble)
Chloé Prunier (IAB, Grenoble)
Jean-Luc Coll (IAB, Grenoble)
Emilie Vassal (CMBA, Grenoble)
Caroline Barette (CMBA, Grenoble)
Catherine Pillet (CMBA, Grenoble)
Emmanuelle Soleilhac (CMBA, Grenoble)
Odile Filhol (INSERM 104, Grenoble)
Attilio di Pietro (UMR5086 CNRS, Lyon)
Charles Dumontet (INSERM 590, Lyon)
Ora Bernard (Victoria, Australia)
Stefan Knapp (Oxford, GB)
Statistics for HTS
Robert Nadon (McGill, Montréal, Canada)
Chemistry
Chi-Hung Nguyen (Institut Curie, Paris)
Claude Lardy (Edelris)
Transfert technology office
Emmanuelle Le Coz (FIST, Paris)
Guillaume Rochet (CNRS, Grenoble)
Michel Ferrand (CEA-Grenoble)
Support for start-up maturation
Frédérique Souq (GRAVIT, Grenoble)
Philippe Ruffin (GRAIN, Grenoble)
CLARA’s Team (2011 Trophy)
Hit to lead & preclinical development
Jean-Jacques Zeiller (CLARA)
Jacques Descotes (CLARA)
Jean-Marc Combette (Solid Drug Development)
Catherine Deloche (Solid Drug Development)
CELLIPSE
Fabrice Paublant (CEO)
Renaud Prudent(COO)
Marc Billaud (SAB member)
Jean-Yves Blay (SAB member)